A novel solvent-free co-grinding preparation improves curcumin bioavailability in healthy volunteers: A single-center crossover study

Curcumin, from the rhizome of turmeric (Curcuma longa L.), has a wide variety of biological activities. Unfortunately, its poor water-solubility greatly limits its bioavailability. The purpose of this study was to evaluate CUMINUP60®, a novel preparation utilizing a solvent-free, co-grinding method designed to improve curcumin’s bioavailability. We performed a single-center crossover experiment to compare the new product with standard 95% curcumin in the blood plasma of twelve healthy adults (10 males, 2 females). Total bioavailability of curcumin and its sulfate and glucuronide conjugates from the test product, measured by their areas under the curve over 12 h (AUC0-T), showed a combined increase of 178-fold over standard curcumin and its conjugates from the reference product. The new product represents a significant improvement for providing greater bioavailability of curcumin, as compared with several other branded preparations. It therefore has broad applications for preparing curcumin as a more effective health ingredient in functional foods, beverages, and nutraceuticals.

The molecular mechanisms behind curcumin's broad spectrum of activities reveal its influence on a wide variety of targets, including multiple transcription factors and a host of enzymes and other gene products [8,13].
Despite its potential benefits, the application of curcumin as a therapeutic agent suffers from its extremely low oral bioavailability. Several strategies have been developed for increasing absorption [14,15,16,17,18,19,20,21,22,23,24,25]. However, their commercial applications can be hampered by overly complicated processing, high cost, low ingredient-loading capacities, gastric irritation, and the necessity of using environmentally unfriendly organic solvents.
To overcome these problems, a simple process was developed using a solvent-free co-grinding method to improve the bioavailability of curcumin with high loading capacity. This preparation boosted cellular bioavailability in three human colon cancer cell lines (Caco-2, HT-29, human Burkitt's lymphoma) and in male Sprague-Dawley rats [26].
In addition to evaluating free curcumin, plasma analyses also followed the concentrations of two extensively transformed curcumin conjugates, glucuronide and sulfate, known in both rodents and in humans [27,28,29]. Results revealed that the preparation manufactured by the new delivery system, CUMINUP60®, improved total bioavailability of curcumin and its conjugates several-fold over that of standard curcumin.
The main aim of the present study was to evaluate the bioavailability and plasma pharmacokinetics of this novel oral delivery system in human subjects in comparison with standard 95% curcumin.

Curcumin study products
The test product used in this study, CUMINUP60®, was developed and supplied by Qingdao Chenland Pharmaceutical Technology Development Co., Ltd. (Qingdao City, Shangdong Province, China). The preparation method was first reported by Ref. [26]. For the current study it entailed combining one part of the excipient, Kolliphor® Poloxamer 407 (BASF, Shanghai, China), with two parts of a turmeric rhizome extract containing 95% curcumin. Two small steel balls were added to the mixture, which was then placed in a grinder for grinding at a frequency of 60 Hz for 2 min (Automatic Grinding Instrument (JXFSTPRP24, Shanghai Jingxin Industrial Development Co., Ltd.).

Human subjects
This study was approved by the Ethics Committee of the National Drug Clinical Trial Institution of Affiliated Hospital of Qingdao University on February 24, 2021, with approval number QYFYEC 2021-014-01. The ethics review and approval process strictly abided by the ethical principles of human medical research in the Declaration of Helsinki, Good Clinical Practice issued by NMPA, ICH/GCP and corresponding requirements of domestic laws and regulations. The study was performed from April 16, 2021, to April 23, 2021, at the National Drug Clinical Trial Institution of Affiliated Hospital of Qingdao University, Shandong Province, China. All volunteers signed the written informed consent form in duplicate, one retained by the study center and the other retained by the subject.
Subjects of both genders were included in the study once they understood the content, processes, and possible adverse reactions from the test product, then signed the informed consent form. Additional inclusion criteria included being able to complete the study as required by the protocol; having taken effective contraception (male and female) within 14 days prior to screening; willing to continue taking contraception voluntarily, without planning a pregnancy within 3 months after the completion of the study; being between the ages of 18-55 years (inclusive); weighing no less than 50 kg (male) or 45 kg (female); and having a BMI between 18.0 and 28.0 (inclusive).
Subjects with the following conditions were excluded from the study: having clinically significant abnormalities as judged by the clinician, including a history of heart, liver, kidney, digestive tract, nervous system, respiratory system, mental disorders, and metabolic disorders; abnormalities based on the physical examination (ECG, clinical laboratory testing, vital signs); testing positive for hepatitis B surface antigen, hepatitis C antibody, HIV antibody, syphilis antibody, or COVID-19 antibody; having a history of specific allergies to pollen, milk or other foods, to two or more drugs, or to curcumin components or analogues; drinking more than 14 units of alcohol per week (1 unit = 285 mL of beer, 25 mL of spirits, or 100 mL of wine); having a history of dysphagia or any gastrointestinal disorder affecting drug absorption (e.g., gastric or small bowel resection, atrophic gastritis, gastrointestinal bleeding, obstruction; being pregnant or lactating; screening positive for drugs, having used drugs in within 3 months prior to the study, or having a history of drug abuse; smoking more than 5 cigarettes per day, on average, within 3 months prior to the study; donating or losing more than 400 mL of blood within 3 months prior to the study, or donating 2 therapeutic doses of platelets (1 therapeutic dose = 12 units of platelets) within 1 month prior to the study; having surgery within 3 months prior to the study; having taken curcumin within 3 months prior to the study; having taken any prescription drug within 14 days prior to the study; having taken any over-the-counter drug, herbal medicine, or other health product within 7 days prior to the study; consuming grapefruit or grapefruit juice, ginger, or curry within 48 h prior to the study; consuming chocolate or any other caffeinated or xanthine-rich drinks or foods (e.g., liver) within 48 h prior to the study undergoing strenuous exercise within 48 h prior to the study; consuming or screening positive for alcohol within 24 h prior to the study. Twelve healthy subjects were enrolled under the protocol (Table 1). Prior to enrollment in the study, all volunteers were made fully aware of the purpose, content and process of the study, the benefits and potential risks of participating in the study, and were informed of that they should participate in the study voluntarily and could be withdrawn from the study at any time and for any reason.
The safety of the test product and the reference product was assessed by the incidences of adverse events, laboratory test results (hematology, urinalysis, and blood biochemistry), vital signs evaluation, ECG results, and physical examination results.
One male subject withdrew before completing the second phase of the study.

Study design
Subjects were evenly divided into two groups. All subjects in both groups began on Day 1 after an overnight fast of at least 10 h. In Period 1, those in one group each received 4 capsules (1600 mg) of the test product. Those in the other group each received 4 capsules (1600 mg) of the reference product. Subjects took their corresponding samples with 240 mL warm water.
Subjects remained in the Clinical Study Center until after blood collections and vital sign examinations were completed and approved through the 72 h period after administration. The potential influence of diet was minimized during the 72-h sampling periods of the study by requiring all subjects to consume the same meals provided by the Clinical Study Center.
Prior to leaving the center, subjects were informed by staff of the washout period precautions and informed to report any adverse events occurring during the washout period. Following a 7-day washout period after the initial administration, subjects returned to the Clinical Study Center for Period 2, beginning on Day 8 of the study. Vital signs were taken again before Period 2 began. Protocols in Period 2 were identical to those of Period 1. The group taking the test product in Period 1 switched to the reference product for Period 2, while the second group switched to the test product. Subjects were allowed to leave the study after completing safety examinations 72 h after initial administration in Period 2.

Plasma sample preparation
Each sample consisted of 4 mL of venous whole blood collected into an anticoagulant tube containing EDTA-K 2 . Samples were collected under a yellow fluorescent lamp throughout the study. Within 30 min of collection, each sample tube was gently inverted 5-6 times and placed in an ice bath before being transferred to a pre-cooled low-temperature centrifuge set at 4 • C. Centrifugation was at 1700×g for 10 min. Aliquots (800 μL) of plasma samples were then put into pre-cooled cryogenic tubes, which were placed within 1 h into a − 20 • C freezer, then transferred within 24 h to a − 80 • C freezer for longer term storage.
Prior to analysis, samples were thawed in wet ice and briefly vortexed before aliquots were transferred to a 96-well deep hole plate.
Aliquots (300 μL) of supernatants were transferred to a 96-well deep hole plate, air-dried with nitrogen, then swirled for 5 min with 150 μL of the formate/MeOH solution and stored at 4 • C prior to analysis.
For curcumin conjugate analyses, 50 μL aliquots were combined with 25 μL of the formate/MeOH solution and eddy mixed for 3 min, followed by 5 min vortexing with 400 μL of 0.1% HCO 2 H in CH 3 CN and 15 min centrifugation at 1700×g at 4 • C.
Aliquots (200 μL) of supernatants were transferred to a 96-well deep hole plate, air-dried with nitrogen, then swirled for 5 min with 200 μL of the formate/MeOH solution and stored at 4 • C prior to analysis.

Analytical methods
Liquid chromatography was performed on a Shimadzu HPLC system (Shimadzu, Tokyo, Japan) consisting of an SCL-10A system controller, two LC-10AD pumps, and an SIL-10AD autosampler. Quantitation was carried out by a Triple Quad 6500 MS/MS system (AB Sciex Pte. Ltd., Singapore) using negative electrospray ionization in multiple-reaction-monitoring (MRM) mode. HPLC analyses were calibrated using curcumin-d 6 as the internal standard.

Pharmacokinetic analysis
Pharmacokinetic parameters were calculated by adopting a noncompartmental model and actual blood sampling time points with SAS V9.4 software. The main parameters were evaluated as times to peak plasma concentration (T max ), maximum plasma concentrations (C max ), and areas under the concentration-time curve (AUC) from time zero to the last sample collection time (T) at which the concentration can be accurately determined (AUC 0-T ).
AUC was determined by the trapezoid rule [30].

Bioavailability analysis
Percent bioavailability (F%) of each constituent was calculated as AUC 0-T from the test product, divided by that of the reference product, x 100. Total bioavailability was determined by calculating F% for the sum of all constituents. Fig. 1 shows concentration curves for curcumin, curcumin sulfate, and curcumin glucuronide in plasma samples over time. Since all substances washed out so quickly, data presentation for graphing the curves was truncated at 12.0 h.

Pharmacokinetics
Concentration of free curcumin from the test product reached a peak at 85 ng/mL ± 0.16 SEM in plasma 1 h after administration (Fig. 1a). Free curcumin from the reference product was not detectable. Concentrations of both the sulfate conjugate (902.36 ng/mL ± 306.98 SEM) and the glucuronide conjugate (10.43 ng/mL ± 3.15 SEM) from the test product peaked at 2 h (Fig. 1b and c). Conjugates from the reference product of sulfate (18.46 ± 10.49 SEM) and glucuronide (0.98 ng/mL ± 0.33 SEM) each reached peak concentration at 5 h. Table 2 lists the bioavailabilities of curcumin and its conjugates in the test product in comparison with the reference product. Calculations were derived by converting ng⋅h/mL from all AUC 0-T data to nmol⋅h/mL.

Product safety
Safety of the test product and the reference product was assessed by the incidences of adverse events, laboratory tests (hematology, urinalysis, and blood biochemistry), vital signs evaluation, ECG results, and physical examination.
Three adverse events occurred in 2 subjects during the first test period after taking the reference product. All adverse events were of Grade 1 [31]. The 3 adverse events were "possibly related" to the reference product. No action was taken. No subjects dropped out of the study due to adverse events.

Table 2
Total bioavailability of curcumin and its conjugates after a single oral administration of the test product vs. the reference product. Notes: F% = (Total AUC 0-T test product/Total AUC 0-T reference product) x 100. Abbreviations: F%, percent bioavailability; AUC area under the curve.
No adverse events were observed after subjects took the test product.
No clinically significant changes in lab tests or vital signs occurred in any subject after administration of either the test product or the reference product.

Discussion
This study confirms the following about the oral administration of 1600 mg of CUMIMUP60® vs. the 95% curcumin reference product: 1) glucuronide and sulfate conjugates predominate in plasma; and, 2) the new formulation vastly improves the bioavailability of curcumin over that of the reference product.
These results also confirm the widely known low absorbability of orally administered free curcumin. The high rate of curcumin conjugation observed here corroborates what has been documented in previous studies [32,33,34,35]. This trend continues even when dosages are much higher, up to 12 g in a single dose [29,36].
BioCurc® is a liquid droplet micellar formulation of curcumin. It has been shown to raise bioavailability of curcumin by 94-fold [45]. BCM-95® is a mixture of turmeric essential oils and curcumin. Its bioavailability was shown to be 6.9-fold greater than that of standard curcumin [46]. CAVACURMIN® consists of curcumin formulated in a γ-cyclodextrin matrix. It was discovered to boost bioavailability of curcumin by 40-fold over a non-formulated curcumin extract. Curcugen® is a preparation of curcumin in a turmeric oleoresin/essential oils matrix. It has been shown to increase relative bioavailability of total curcuminoids by 49.5-fold [48]. Curcumin C3 Complex® is a standardized curcuminoid extract combined with piperine (Bioperine®). The most recently reported relative bioavailability of curcumins combined with piperine was comparatively low at 1.1-fold [49]. This level contrasts with results of an earlier study showing a curcumin-piperine combination to boost relative bioavailability by 20-fold [21]. CurcuRouge® is made from melted standard curcumin that has been coarsely ground and mixed with modified starch. Although its relative bioavailability has not been directly evaluated, the 91.8-fold increase cited in Table 3 derives from a comparison study with Theracurmin® [50], whose relative bioavailability has been reported [22]. CurcuWin® consists of curcumin enhanced for water-solubility with the hydrophilic carrier, polyvinyl pyrrolidone. It has shown a 49.5-fold increase in relative bioavailability over standard curcumin [51]. Cureit® was developed by formulating a curcumin complex with a polar-non-polar sandwich technology [57]. It has boosted curcumin bioavailability by 10-fold [52]. CurQfen® is a combination of curcumin and a galactomannan-rich fiber extract from fenugreek seeds. It has been shown to boost curcumin bioavailability by 45.5-fold [53]. Longvida® uses particle-based technology for preparing curcumin with a mixture of lecithin and phospholipids from soy. It has been shown to boost curcumin bioavailability by 100-fold [54]. Meriva® is a curcumin-phosphatidylcholine complex made with soy lecithin. It has been shown to boost curcumin bioavailablity by 29-fold over standard curcumin [55]. MicroActive® Curcumin SR is a water dispersible formulation containing dispersed micronized curcuminoids in a sustained-release matrix. It has been shown to increase curcumin bioavailability by 9.7-fold [19]. NovaSOL® is a micelle preparation made with a nonionic surfactant, Tween-80. This formulation has boosted curcumin bioavailability 185-fold [42]. Ther-acurmin® was prepared by homogenizing a gum ghatti polysaccharide-rich extract with an alcohol-extracted curcumin powder. This preparation has enhanced curcumin bioavailability by 27-fold [22]. TurmiPure Gold® is a mixture of turmeric, acacia gum, and sunflower oil, emulsified with soapbark (quillaja) extract. It was recently found to increase relative bioavailability of curcumin by 24.2-fold over standard curcumin [49]. TurmXTRA® 60 N is a proprietary water-dispersible turmeric extract. It has recently been shown to provide a 10-fold boost in bioavailability of curcumin [56]. As these data show, during the past decade researchers have created different formulations with a focus on improving the bioavailability of curcumin for commercialization. As a result, different products reveal a varying range of enhanced bioavailabilities. Few exact comparisons have been published for head-to-head evaluations within the same study [37,39,47,49,50], [58,59,60]. They have been limited to comparing 2-4 products at a time. For the majority of products, comparisons of bioavailabilities among different studies are inexact due to varying study designs, analytical techniques, pharmacokinetic parameters, dosages, and other relevant details of each formulation. In spite of these drawbacks, literature reports reveal clear trends, with several products exhibiting over 100-fold higher bioavailability relative to standard unformulated curcumin [42,54,61] or close it [45,50].
As of this study, we can now add CUMINUP60® to the list of high-performing curcumin formulations. In addition, the extra advantage of the preparation reported here is that it doesn't involve toxic solvents [26]. As previously determined, this preparation also has a high drug-loading capacity of 65.5%, which improves its aqueous solubility. Furthermore, the solvent-free co-grinding mixture is simple enough to enhance its suitability for industrial production.
Although the small size of this study precludes any formal safety end point analysis or statistical certainty of safety, the safety profile observed here is consistent with previous clinical and preclinical data [32,35,36,46,62]. However, oral administration of both the test product and the reference product were found to be safe for humans under fasting conditions.
Overcoming the barrier of curcumin's poor bioavailability is a prerequisite for developing effective functional foods and beverages, supplements, and pharmaceuticals [63,64]. Aside from an earlier study of an unbranded beverage containing Theracumin® [65], food, beverage, and supplement manufacturers have not yet taken full advantage of curcumin formulations with well-documented enhanced bioavailabilities. At least a half-dozen such formulations, including CUMINUP60®, hold great promise for future developments of such over-the-counter health consumables [66].
In addition, although free curcumin is widely acknowledged as the biologically active form of curcumin, many studies have begun to focus on the potential roles of Phase II metabolites such as its glucuronide conjugate [67]. For example, curcumin glucuronide has shown potential for direct anti-cancer activity [68] and for the treatment effects of curcumin in vivo mediated at NF-κB [67]. Such roles seem to depend on deconjugation to keep free curcumin in blood [69] and bone [70]. These findings may help explain turmeric's traditional use in reducing inflammation in tissues rich in the β-glucuronidase that drives deconjugation.
Limitations of the current study point to two main areas where future research can bolster what we know about curcumin bioavailability. The first limitation is the lack of determination of additional derivatives of curcumin, including demethoxycurcuin, bisdemethoxycurcumin, dihydrocurcumin, tetrahydrocurcumin, and hexahydrocurcumin. Although these metabolites are not determined in most studies, a need exists for evaluating their levels in plasma in order to better understand the relationships between curcuminoid pharmacokinetics and physiological/pharmacological effects.
The second limitation is not evaluating the effects of the enhanced absorption of curcumin in CUMINUP60® regarding different physiological outcomes. Comparing treatment effects between free curcumin and CUMINUP60® would fulfill that need.

Conclusions
Curcumin is one of the most investigated natural products. Its health benefits have been well established. Advances in the drug delivery field hold promising strategies for increasing its bioavailability [44]. The results of the present study provide a new strategy building on that promise. We suggest the co-grinding, solvent-free process used here has broad applications for preparing curcumin as a more effective health ingredient in functional foods and beverages and nutraceuticals.

Data availability
The datasets analyzed during the current study are available from the corresponding author upon reasonable request.

Declaration of competing interest
The authors declare the following financial interests/personal relationships which may be considered as a potential competing interest: CUMINUP60® is the registered trademark name of Chenland Nutritionals, Inc., for the test product used in the present study. Chenland Nutritionals, Inc., had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.